Nicotinamide adenine dinucleotide (NAD) is an essential molecule in cells. In addition to its role in oxidation-reduction reactions, in which NAD(H) and its phosphorylated form, NADP(H), act as hydride donors and acceptors, NAD is also important for other cellular processes, such as the activity of NAD-dependent DNA ligases, mono and poly ADP-ribosylation of proteins, and production of the intracellular calcium-mobilizing molecules cADPR and NaADP (1), (2).
NAD is synthesized via a multi-step de novo pathway or via a pyridine salvage pathway. The enzyme nicotinic acid mononucleotide adenylyl transferase (NaMN AT, EC 2.7.7.18) sits at the convergence of these two pathways. NaMN AT catalyzes the conversion of ATP and nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) (FIG. 1), which is directly processed to NAD by NAD synthetase. The nadD gene, encoding NaMN AT, was the first enzyme demonstrated to be essential of NAD biosynthesis by both the de novo and salvage pathways (3). A number of enzymes demonstrating in vitro adenylyltransferase activity for NaMN and NMN have been identified in eukarya, archaea and bacteria (4), (5), (6), (7), (8), (9), (10), (11). Along with sequence homology, the specificity of these enzymes for NMN versus NaMN provides a useful method for classifying new genes within this family.
While there is sequence conservation between the eubacterial nadD genes (FIG. 2), sequence alignment of nadD NaMN ATs to the eukaryotic enzymes or archeal enzymes is difficult outside of the region surrounding the (H/T)XGH (SEQ ID NO:18) nucleotidyl transferase consensus sequence. Adenylyltransferases encoded by the nadD gene prefer the nicotinic acid containing NaMN over NMN as a substrate by a factor that ranges from 6–1 to 2000–1 (12), (13), (4). Eubacteria also contain enzymes that demonstrate higher specificity for the nicotinamide containing NMN. This group the products of the nadR gene, which in addition to its regulatory role in NAD biosynthesis, also contains NMN AT activity (14). The eukaryotic and archeal NMN AT (EC 2.7.7.1), such as those from human (15), Methanococcus jannaschii (16) and Methanobacterium thermoautotrophicum (17), either demonstrate higher specificity for NMN as a substrate, as compared to NaMN, or show little preference for either substrate (4).
Primary sequence studies indicate that NaMN AT belongs to the nucleotidyltransferase α/β phosphodiesterases superfamily of enzymes that contain the (H/T)XGH (SEQ ID NO:18) signature motif. Members of this family share the same basic catalytic mechanism, involving direct nucleophilic attack upon an α-phosphate followed by the release of pyrophosphate, while the enzyme provides stabilization of the transition state prior to the formation of a new phosphodiester bond. The recent structure determination of NMN ATs, from Methanococcus jannaschii and Methanobacterium thermoautotrophicum, has allowed this sequence and functional homology to be extended to the structural conservation of residues involved in substrate binding and catalysis (16), (17).
Genes that have been identified to be essential for bacterial survival are currently being evaluated for their potential as targets for anti-microbial chemotherapy. Understanding the biochemical, physical and structural properties of these essential enzymes and placing them in a larger biological context are the first steps in exploring this potential. The present invention is based on the identification of an unassigned reading frame in B. subtilis (yqeJ) as a NaMN AT. The recombinant enzyme was expressed in E. coli and shown to prefer NaMN as a substrate to NMN, allowing the assignment of it as the nadD gene of B. subtilis. It differs from the NMN ATs from Metanococcus jannaschii and Methanobacterium thermoautotrophicum both in its substrate specificity and oligomeric state. It is homodimeric as opposed to a homo-hexamer (16), (17). The three dimensional structure of NaMN AT from B. subtilis has been determined to 2.2 Å and 3.2 Å with the NaAD bound. This has allowed the identification of key residues in substrate binding and catalysis. These structures will provide invaluable information in the ongoing development of anti-microbial agents targeting NAD biosynthesis.